2015 In the last year, our work has covered several major areas: I. Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 15 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength. We helped describe their roles in TCR-induced activation of PLC-g and Ca++ mobilization, defined kinase-independent functions of Itk in regulating the actin cytoskeleton and cell adhesion, (required for effector T cell functions). Confirming its importance, mutations of Itk have now been described in a profound EBV-induced lethal immunodeficiency. Recently, we have focused on the effects of mutations on patterns of cytokine production by CD4+ T helper cells and the ability of mice to respond to distinct types of infection. We recently published work demonstrating that Itk helps regulate the balance between Th17 cells, CD4+ effector T cells that respond to extracellular bacteria (but which also contribute to autoimmune and inflammatory disorders), and regulatory T cells (Tregs), a subset of cells responsible for keeping immune responses in check. Our work provided evidence that Itk is part of a positive feedback loop that normally represses expression of the lipid phosphatase Pten, upon TCR activation, thereby affecting T cell responses to multiple signaling pathways and suggesting Itk as a potential therapeutic target for autoimmunity (Gomez-Rodriguez et al, J. Exp Med 2014). In the last year, we have examined the role of Itk in the development of Th9 cells, important contributors to asthma and airway hypersensitivity. Previous work had demonstrated that Itk was required for Type II immune responses that lead to asthma. We have now shown that Itk is strictly required for the expression of IL-9 and link this defect to TCR signaling defects. We further show that IL-2 can rescue multiple defects in Itk-deficient T cells, including the induction of IRF4, a transcription factor linked to TCR signaling strength. Our work provides insight into mechanisms by which IL-2 promotes the function of sub-optimally activated T cells and suggests Itk as a therapeutic target for IL-9-mediated diseases (Gomez-Rodriguez et al, submitted). Furthermore, as an expert in T helper differentiation and in gene-targeting, Dr. Gomez-Rodriguez has contributed to the work of several other laboratories. II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific member of the PI3K catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014). We have recently found that although patient cells show normal to increased killing of P815 murine mastocytoma cells coated with anti-CD3 antibodies, a measure of general cytotoxicity, P110&#61540;elta CD8 cells are unable to efficiently kill autologous EBV-infected target B cells and link this to altered expression of PDL1. To further understand these defects, we have generated a mouse model and are using these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. III. SAP and the regulation of Tfh cells and humoral immune responses: Another major focus of our work is SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1), characterized by fatal EBV-infection, lymphomas, and antibody defects. SAP is a small SH2 containing adaptor that binds phosphorylated tyrosine residues in the intracellular tails of SLAM family co-stimulatory receptors. We previously generated SAP-deficient mice and found these mice recapitulate features of XLP, including increased T cell activation and decreased antibody production upon infection (Czar et al PNAS). Notably, SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination. These defects have been confirmed in humans with XLP. We showed that SAP-/- T cells have a selective defect inn adhesion to B cells but not other cells, preventing them from delivering contact-dependent signals required for B cells to form germinal centers (Qi et al, Nature, 2008; Cannons et al, Immunity, 2010), leading us to propose that defective T cell help for B cell antibody responses and defective T and NK cell killing of EBV-infected B cells in XLP, resulted from impaired interactions with B cells (Schwartzberg, Nat Rev. Immunol 2009). Confirming this idea, we found that SAP-/- CD8 cytotoxic lymphocytes show selective defects in killing B cell targets, despite normal killing of other cells (Zhao et al 2012, Immunity) resulting from a strong negative signal that alters the immunological synapse formed between T cells and B cell targets, thereby inhibiting cell interactions and cytolysis. Furthermore, we found similar results in CD4 cells, (Kageyama et al, 2012 Immunity) providing common mechanistic insight into the pathophysiology of XLP and suggesting potential therapeutic approaches to XLP via blocking SLAM family members. Our work further provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells, the critical helper T cell population required for providing signals to B cells for germinal center formation and long-term humoral immunity, the heart of protective responses to most immunizations. In the last year, we have continued our studies of this important T cell population. Using RNAseq to evaluation Tfh-specific gene-expression signatures, we found that the transcription factor TCF1, a component of the Wnt signaling pathway, is selectively expressed in Tfh cells in response to viral infection. Using conditional knockout mice and shRNA knockouts, we and others recently provided evidence that TCF1 is required for Tfh responses to viral infection (Wu et al, Cell Reports, In Press). Our work helps provide insight into the regulation of this important T helper cell population, which permits an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013).